The long term objective of the proposed research is 1) to understand the molecular basis of tropomyosin-troponin linked regulation in vertebrate skeletal muscle and 2) to determine if a thin filament regulatory system involving tropomyosin exists in nonmuscle cells using human blood platelets as an experimental system. We wish to relate the functional interactions of tropomyosin and tropomyosin-binding proteins with actin to cell movement and shape. As the first step towards defining a thin filament regulatory system in platelets we have characterized a protein (TMBP) distinct from troponin that binds to tropomyosin. The binding of TMBP to tropomyosin is influenced by divalent cations, Ca2+ and Mg2+, suggestive of a regulatory role. In the present proposal we will use immunological methods to learn if TMBP is present in other tissues and cells and if it is associated with stress fibers in cultured cells. We will also correlate its association with actin and tropomyosin to the state of activation of the platelet. We will characterize its interaction with purified actin and tropomyosin by studying the effects on actin polymerization, the binding by cosedimentation, and the effect on the actin activated ATPase by myosin. Through these experiments we hope to learn if TMBP and tropomyosin are regulatory proteins in blood platelets. To further define the structure of the thin filament, we will develop topographical maps of tropomyosin, tropomyosin-troponin, and of the regulatory proteins complexed with actin with the aim of describing the surface structure of the proteins and of learning which regions of each protein are involved in interactions with each other. Lysine will be used as a probe for the relative accessibility of different regions of the surface of the protein using a competitive labeling procedure with acetic anhydride. We will relate the reactivities of the tropomyosin lysines to the proposed coiled-coil structure and learn if troponin binds to the predicted binding sites at Cys 190 and the overlap region. We will also test predictions of the steric blocking model.